The proposed research project extends previous work from our laboratory and is aimed at gaining a complete functional and structural characterization of thrombin allostery and how it influences recognition of physiologic substrates, effectors and inhibitors. We will use kinetic and site-directed mutagenesis studies of the hydrophobic rim, the W60d loop and the allosteric switch to complete the identification of the functional epitopes of thrombin recognizing fibrinogen, fibrin, PAR1, PAR3, PAR4, protein C, thrombomodulin and antithrombin III (specific aim 1). The contribution of thrombin residues to substrate recognition in the two allosteric conformations of the enzyme, slow and fast, will be elucidated in unprecedented detail. The new information to emerge from the proposed studies will broaden our understanding of thrombin interactions in the blood, which will enable in the long run a better pharmacological control of its functions in vivo. The functional studies will be complemented by detailed X-ray crystallographic analysis of the allosteric conformations of thrombin free and bound to active site inhibitors (specific aim 2). Mutants of residues located within the allosteric switch of the enzyme and directly involved in substrate recognition will also be crystallized in the free and bound forms. This highly integrated structure-function approach to the study of thrombin-substrate interactions will allow us to test basic determinants of substrate recognition by proteases in general. The relevance of the proposed studies will therefore extend to other allosteric proteases involved in blood coagulation and to the whole family of serine proteases to which thrombin belongs. The substantial new knowledge to be generated from the proposed studies will also be used to rationally engineer thrombin mutants with enhanced specificity toward the anticoagulant protein C (specific aim 3). These protein-engineering studies will provide significant advances toward the design of new proteases with desired activity and specificity that can find pharmacological and biotechnological applications.